Exploring and dissecting genome-wide transcriptional responses of Penicillium chrysogenum to phenylacetic acid

In studies on beta-lactam production by Penicillium chrysogenum, addition and omission of a side-chain precursor is commonly used to generate producing and non-producing scenarios. To dissect effects of penicillin-G production and of its side-chain precursor phenylacetic acid (PAA), a derivative of a penicillin-G high-producing strain without a functional penicillin-biosynthesis gene cluster (pcbAB-pcbC-penDE) was constructed. The copy number of this cluster was first reduced to one via spontaneous recombination. The remaining copy was removed by targeted deletion, thereby completely abolishing beta-lactam biosynthesis. In glucose-limited chemostat cultures of the high-producing and cluster-free strains, PAA addition caused a small reduction of the biomass yield, consistent with PAA acting as a weak-organic-acid uncoupler. A low rate of penicillin-G-independent PAA consumption indicated activity of a PAA-degrading pathway. Microarray-based analysis on chemostat cultures of the high-producing and cluster-free strains, grown in the presence and absence of PAA, showed that: (i) Absence of a penicillin gene cluster resulted in transcriptional upregulation of a gene cluster putatively involved in production of the secondary metabolite aristolochene and its derivatives, (ii) The homogentisate pathway for PAA catabolism is strongly transcriptionally upregulated in PAA-supplemented cultures (iii) Several genes involved in nitrogen and sulfur metabolism were transcriptionally upregulated under penicillin-G producing conditions only, suggesting a drain of amino-acid precursor pools. Furthermore, the number of candidate genes for penicillin transporters was strongly reduced, thus enabling a focusing of functional analysis studies. This study demonstrates the usefulness of combinatorial transcriptome analysis in chemostat cultures to dissect effects of biological and process parameters on transcriptional regulation. Mutants of P. chrysogenum impaired in penicillin biosynthesis have been described. Most of these mutants were derived by random mutagenesis from the low-producing strain Wisconsin54-1255, which contains one copy of the penicillin biosynthesis cluster. Although these mutants were very useful for studying gene expression and gene/enzyme relationships, for identification of the factors important for penicillin-G production and PAA consumption a strain obtained from a high-producing strain background by targeted deletion is more beneficial. To this end we constructed a strain in which the tandem-repeated penicillin biosynthesis cluster was specifically deleted, whereas the strain background was retained. In such a way it is possible to identify, by a combinatorial approach, those genes affected by phenylacetic acid and those specifically important for penicillin-G biosynthesis.